Ammonia is a neurotoxin that predominantly affects astrocytes. Disturbed mitochondrial function and oxidative stress, factors implicated in the induction of the mitochondrial permeability transition (MPT), appear to be involved in the mechanism of ammonia neurotoxicity. We have recently shown that ammonia induces the MPT in cultured astrocytes. To elucidate the mechanisms of the MPT, we examined the role of oxidative stress and glutamine, a byproduct of ammonia metabolism. The ammonia-induced MPT was blocked by antioxidants, suggesting a causal role of oxidative stress. Direct application of glutamine (4.5-7.0 mM) to cultured astrocytes increased free radical production and induced the MPT. Treatment of astrocytes with the mitochondrial glutaminase inhibitor, 6-diazo-5-oxo-L-norleucine, completely blocked free radical formation and the MPT, suggesting that high ammonia concentrations in mitochondria resulting from glutamine hydrolysis may be responsible for the effects of glutamine. These studies suggest that oxidative stress and glutamine play major roles in the induction of the MPT associated with ammonia neurotoxicity.